1. Field of the Invention
The present invention relates to a mutant firefly luciferase for which the proportion of activity induced by dATP to activity induced by ATP (dATP/ATP) is lower than such proportion for the wild-type firefly luciferase, the gene thereof, a recombinant vector comprising the gene, and a method for evaluating the activity of the mutant firefly luciferase.
2. Background Art
Sequencing methods based on the Sanger's sequencing method have been widely used as sequencing methods whereby a DNA nucleotide sequence is determined. In such a method, a primer is allowed to bind to a DNA template and a reaction for synthesizing new DNA is carried out by incorporating deoxyribonucleotides (dNTPs: dATP, dGTP, dCTP, and dTTP) with the use of a DNA polymerase, provided that the 3′ end of the primer is designated as a starting point. In such case, small amounts of dideoxyribonucleotides (ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP) that have been labeled with four different phosphors are added to a reaction system in a preliminary step. When ddNTPs are incorporated upon reaction, the synthesis reaction is terminated, resulting in generation of DNA fragments in different sizes. Such products contain different phosphors depending on the types of incorporated ddNTP. Thus, the nucleotide sequence of the template DNA can be determined by denaturing such a product into a single strand, followed by electrophoresis and size fractionation.
In the genome project for analyzing the total human DNA nucleotide sequence, sequence analysis was carried out by such a method based on the Sanger's sequencing method. In the project, a sequence analysis apparatus using capillary electrophoresis was newly introduced. Thus, automatic and high-speed analysis has been realized and thus it has become possible to analyze a number of DNA nucleotide sequences.
In recent years, sequence analysis methods based on different principles have been actively developed in order to analyze an increased number of DNA nucleotide sequences at low cost in a rapid manner. For instance, 454 Life Sciences has developed massively parallel high-speed sequencing technology using a system for simultaneously analyzing a number of nucleotide sequences by carrying out a sequence reaction on beads placed on flow cells, and it has already marketed such products.
A method based on principles for sequencing methods used for the massively parallel high-speed sequencing technology is a DNA nucleotide sequence analysis method using bioluminescence, which is also referred to as a pyrosequencing method. In this method, a primer is allowed to bind to a DNA template, 4 types of dNTPs are sequentially added thereto, and then an elongation reaction with the use of a DNA polymerase is carried out. In such case, when a dNTP matched to the template is added, an elongation reaction takes place, resulting in generation of pyrophosphoric acid (PPi). The thus generated PPi is converted into ATP by ATP sulfurylase or the like. Further, the obtained ATP is used as a substrate such that a luciferase induces a luminescent reaction. The occurrence of luminescence indicates incorporation of a matched dNTP, and thus the nucleotide sequence of the template can be determined.
Meanwhile, a luciferase that is used as an enzyme in the above reaction system has a unique characteristic of catalyzing a luminescent reaction. Thus, luciferases are used in a variety of measurement systems involving bacterial count based on ATP quantification, cell proliferation assay, reporter assay for measuring gene transfer activity, intracellular marker/enzyme high sensitive assay, and the like, in addition to sequence analysis. Further, luminescent reactions caused by luciferases can be used at cell, culture tissue, and individual levels. Thus, luciferases are industrially important enzymes that are essential for the field of luminescence imaging.
Thus, in order to achieve further industrial applications of luciferases, a variety of modified luciferases have been developed. For instance, a luciferase having an increased luminescence intensity as a result of substitution in the amino acid sequence has been reported (JP Patent Publication (Kokai) No. 2007-97577 A). It is described that at least one amino acid among the 419th to 428th amino acids is substituted with a non-polar amino acid (alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, or tryptophan) having a molecular weight larger than that of the amino acid such that the luminescence intensity is increased.
In addition to the above, the following mutant luciferases have been reported: mutant luciferases having improved thermal stability (JP Patent No. 3048466, JP Patent Publication (Kokai) No. 2000-197487 A, JP Patent Publication (Kohyo) No. 9-510610 A (1997), and JP Patent Publication (Kohyo) No. 2003-518912 A); a mutant luciferase resistant to a surfactant (JP Patent Publication (Kokai) No. 11-239493 A (1999)); mutant luciferases having improved substrate affinity (WO99/02697, JP Patent Publication (Kohyo) No. 10-512750 A (1998), and JP Patent Publication (Kohyo) No. 2001-518799 A); mutant luciferases having different luminescence wavelengths (JP Patent No. 2666561 and JP Patent Publication (Kohyo) No. 2003-512071 A); and a mutant luciferase in which luminescence is maintained at a high level (JP Patent Publication (Kokai) No. 2000-197484 A).